Mitochondria provide the energy used by every cell and may be central to aging and its associated diseases. The side products of mitochondrial metabolism include reactive oxygen species which have been closely associated with aging related damage. A family of genes called the sirtuins exists in nearly every life form and has been closely associated with aging regulation. In humans, three members of this gene family make products that localize to the mitochondria. Of these, the gene SIRT3 has been specifically linked with long lifespan in humans. Preliminary results from our laboratory indicate that SIRT3 protein may function by altering mitochondrial production of reactive oxygen species and/or altering the cell's ability to adapt to these ions, which may be damaging if improperly regulated or present in inappropriate concentration. These data indicate that the deacetylase activity of SIRT3 is critical for this function. To further define the mechanism by which SIRT3 alters the cellular response to oxidative stress, we propose to demonstrate additional substrates this enzyme acts on and how this results in altered cellular function. Our specific aims include: 1) determining the set of protein substrates deacetylated by SIRT3 inside the mitochondria, and 2) demonstrating the effect of altered acetylation on substrate enzymatic activity. To achieve these goals we will use bioinformatic and proteomic techniques to generate candidate substrates, which we will validate in vitro using biochemical analysis. This proposal is highly relevant to public health and to the mission of NIA since sirtuins are strongly linked to lifespan regulation in a variety of model organisms. Investigations into pathways activated by this protein promise to elucidate basic biochemical mechanisms underlying aging on a cellular level. Additionally, other pathological processes such as those triggered by obesity may involve dysregulation of these pathways and this broadens the public health impact of this work. PUBLIC HEALTH RELEVANCE: This proposal will try to discover the biochemical action of a protein named SIRT3. This protein comes from a gene which has been linked to long lifespan and control of metabolism. Understanding its biochemical action may potentially help us understand the normal aging process and diseases associated with aging, for example diabetes or cancer.